Mutations at the rug5 ( rug osus 5 ) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B 2 and B 3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B 2 and B 3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed. INTRODUCTIONThe starch synthases that catalyze the synthesis of the branched amylopectin component of the starch granule are poorly understood. It is well established that a specific class of granule-bound starch synthases (known as granulebound starch synthase I or GBSSI) is responsible for the synthesis of the unbranched amylose component of the granule. Two or more distinct isoforms other than GBSSI are present in storage organs of the species examined to date, and these together with starch branching enzymes (SBEs) are responsible for the synthesis of amylopectin. However, it is not clear whether different isoforms play qualitatively distinct roles in amylopectin synthesis . Study of the roles of such isoforms is hampered by a lack of mutations that affect them specifically and exclusively. Analysis of transgenic potato plants in which activities of specific isoforms have been reduced (Edwards et al., 1995;Abel et al., 1996;Marshall et al., 1996) has thus far not revealed whether these isoforms have qualitatively distinct roles.In this study, we elucidate the role of the major isoform of starch synthase present in the soluble fraction of the developing pea embryo. Starch synthase II (SSII) is a protein of 77 kD that accounts for 60 to 70% of the soluble starch synthase activity of the pea embryo. It is also present within the matrix of the starch granule (Smith, 1990;Denyer and Smith, 1992; Dry et al., 1992; Denyer et al., 1993;Edwards et al., 1996). Analysis of mutant lines of peas from which GBSSI is absent has shown conclusively that SSII is not involved in amylose synthesis (Denyer et al., 1995a). It is reasonable to assume, therefore, that SSII is important in the synthesis of amylopectin.Amylopectin is a highly branched polymer consisting of linear chains of ␣ (1,4)-linked glucose residues joined together by ␣ (1,6)-linkages. Within the granule, the chains are thought to be arranged in clusters at intervals of 9 nm, within which chains associate t...
Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations.
Mutations at the rug5 ( rug osus 5 ) locus have been used to elucidate the role of the major soluble isoform of starch synthase II (SSII) in amylopectin synthesis in the developing pea embryo. The SSII gene maps to the rug5 locus, and the gene in one of three rug5 mutant lines has been shown to carry a base pair substitution that introduces a stop codon into the open reading frame. All three mutant alleles cause a dramatic reduction or loss of the SSII protein. The mutations have pleiotropic effects on the activities of other isoforms of starch synthase but apparently not on those of other enzymes of starch synthesis. These mutations result in abnormal starch granule morphology and amylopectin structure. Amylopectin contains fewer chains of intermediate length (B 2 and B 3 chains) and more very short and very long chains than does amylopectin from wild-type embryos. The results suggest that SSII may play a specific role in the synthesis of B 2 and B 3 chains of amylopectin. The extent to which these findings can be extrapolated to other species is discussed. INTRODUCTIONThe starch synthases that catalyze the synthesis of the branched amylopectin component of the starch granule are poorly understood. It is well established that a specific class of granule-bound starch synthases (known as granulebound starch synthase I or GBSSI) is responsible for the synthesis of the unbranched amylose component of the granule. Two or more distinct isoforms other than GBSSI are present in storage organs of the species examined to date, and these together with starch branching enzymes (SBEs) are responsible for the synthesis of amylopectin. However, it is not clear whether different isoforms play qualitatively distinct roles in amylopectin synthesis . Study of the roles of such isoforms is hampered by a lack of mutations that affect them specifically and exclusively. Analysis of transgenic potato plants in which activities of specific isoforms have been reduced (Edwards et al., 1995;Abel et al., 1996;Marshall et al., 1996) has thus far not revealed whether these isoforms have qualitatively distinct roles.In this study, we elucidate the role of the major isoform of starch synthase present in the soluble fraction of the developing pea embryo. Starch synthase II (SSII) is a protein of 77 kD that accounts for 60 to 70% of the soluble starch synthase activity of the pea embryo. It is also present within the matrix of the starch granule (Smith, 1990;Denyer and Smith, 1992; Dry et al., 1992; Denyer et al., 1993;Edwards et al., 1996). Analysis of mutant lines of peas from which GBSSI is absent has shown conclusively that SSII is not involved in amylose synthesis (Denyer et al., 1995a). It is reasonable to assume, therefore, that SSII is important in the synthesis of amylopectin.Amylopectin is a highly branched polymer consisting of linear chains of ␣ (1,4)-linked glucose residues joined together by ␣ (1,6)-linkages. Within the granule, the chains are thought to be arranged in clusters at intervals of 9 nm, within which chains associate t...
SummaryThe impact of a mutation at the r locus of peas (Pisum sativum L.) on the structure of starch in the leaf has been studied. The mutation specifically eliminates the A class of isoform of starch-branching enzyme (SBE A) from the leaf, causing a 10-fold reduction in the total activity of the enzyme. Gel-permeation chromatography and thymol precipitation show that wild-type leaf starch consists of polymers with the general characteristics of amylose and amylopectin, although amylose is only a very minor component of the starch. High-performance anion exchange chromatography (HPAEC) of debranched amylopectin reveals that the distribution profile of branch lengths is strongly polymodal, and distinctly different from that of the amylopectin of storage starches. The mutation at the r locus results in the appearance of an amylopectinlike glucan of low molecular weight in the starch. The absorbance of the iodine complex of the amylopectin and analysis by HPAEC both indicate that the mutation causes an increase in the average branch length of the amylopectin but does not affect the polymodal nature of the distribution of branch lengths. The extent to which these effects of the mutation are specifically due to the loss of SBE A is discussed. It is suggested that differences in properties between isoforms of SBE are not the main factors that determine the polymodal distribution of branch lengths in amylopectin.
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